Epstein-Barr Virus-Induced Gene-3 Is Expressed in Human Atheroma Plaques
2009; Elsevier BV; Volume: 175; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2009.080752
ISSN1525-2191
AutoresSybille Kempe, Philipp Heinz, Enikö Kókai, Odile Devergne, Nikolaus Marx, Thomas Wirth,
Tópico(s)Immune Cell Function and Interaction
ResumoAtherosclerosis is characterized by a complex immune response in the vessel wall, involving both inflammation and autoimmune processes. Epstein-Barr virus-induced gene 3 (Ebi3) is a member of the interleukin (IL)-12 heterodimeric cytokine family, which has important immunomodulatory functions. To date, little is known about the role of Ebi3 in vascular disease. We examined the expression of Ebi3 in human atheromatous lesions and analyzed its transcriptional regulation in vascular cells. The in situ expression of Ebi3 in human endarterectomy specimens was analyzed by immunohistochemistry. In these lesions, smooth muscle cells expressed Ebi3 as well as the IL-27α/p28 and IL-12α/p35 subunits. Primary aortic smooth muscle cells up-regulated Ebi3 in response to proinflammatory stimuli like tumor necrosis factor-α and interferon-γ. Interestingly, pretreatment of these cells with the peroxisome proliferator-activated receptor-γ agonist rosiglitazone strongly reduced Ebi3 induction. Chromatin immunoprecipitation experiments revealed that this inhibition is due to interference with p65/RelA recruitment to the Ebi3 promoter. Our data support a possible role of Ebi3 in atherogenesis either as homodimer or as IL-27/IL-35 heterodimer, and suggest that Ebi3 could be an interesting target for therapeutic manipulation in atherosclerosis. Atherosclerosis is characterized by a complex immune response in the vessel wall, involving both inflammation and autoimmune processes. Epstein-Barr virus-induced gene 3 (Ebi3) is a member of the interleukin (IL)-12 heterodimeric cytokine family, which has important immunomodulatory functions. To date, little is known about the role of Ebi3 in vascular disease. We examined the expression of Ebi3 in human atheromatous lesions and analyzed its transcriptional regulation in vascular cells. The in situ expression of Ebi3 in human endarterectomy specimens was analyzed by immunohistochemistry. In these lesions, smooth muscle cells expressed Ebi3 as well as the IL-27α/p28 and IL-12α/p35 subunits. Primary aortic smooth muscle cells up-regulated Ebi3 in response to proinflammatory stimuli like tumor necrosis factor-α and interferon-γ. Interestingly, pretreatment of these cells with the peroxisome proliferator-activated receptor-γ agonist rosiglitazone strongly reduced Ebi3 induction. Chromatin immunoprecipitation experiments revealed that this inhibition is due to interference with p65/RelA recruitment to the Ebi3 promoter. Our data support a possible role of Ebi3 in atherogenesis either as homodimer or as IL-27/IL-35 heterodimer, and suggest that Ebi3 could be an interesting target for therapeutic manipulation in atherosclerosis. Atherosclerosis is an inflammatory disease of the arterial walls with a complex etiology and in which the immune system plays a major role.1Hansson GK Inflammation, atherosclerosis, and coronary artery disease.N Engl J Med. 2005; 352: 1685-1695Crossref PubMed Scopus (6912) Google Scholar Atheroma lesions are characterized by the accumulation of lipid particles and immune cells in subendothelial regions, leading to plaque formation and to narrowing of the arterial lumen. Plaque rupture, can result in thrombosis, the gravest clinical complication of atherosclerosis.2Libby P Aikawa M Stabilization of atherosclerotic plaques: new mechanisms and clinical targets.Nat Med. 2002; 8: 1257-1262Crossref PubMed Scopus (497) Google Scholar, 3Hallenbeck JM Hansson GK Becker KJ Immunology of ischemic vascular disease: plaque to attack.Trends Immunol. 2005; 26: 550-556Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar Activated macrophages and T cells are the main immune components in these lesions.4Jonasson L Holm J Skalli O Bondjers G Hansson GK Regional accumulations of T cells, macrophages, and smooth muscle cells in the human atherosclerotic plaque.Arteriosclerosis. 1986; 6: 131-138Crossref PubMed Google Scholar T lymphocytes constitute approximately 10% to 20% of the cell population in advanced human plaques and they often congregate at sites of atheroma rupture.5Emeson EE Shen ML Bell CG Qureshi A Inhibition of atherosclerosis in CD4 T-cell-ablated and nude (nu/nu) C57BL/6 hyperlipidemic mice.Am J Pathol. 1996; 149: 675-685PubMed Google Scholar, 6Zhou X Nicoletti A Elhage R Hansson GK Transfer of CD4(+) T cells aggravates atherosclerosis in immunodeficient apolipoprotein E knockout mice.Circulation. 2000; 102: 2919-2922Crossref PubMed Scopus (485) Google Scholar This cell population exhibits a type 1 T helper (TH1) cell phenotype.4Jonasson L Holm J Skalli O Bondjers G Hansson GK Regional accumulations of T cells, macrophages, and smooth muscle cells in the human atherosclerotic plaque.Arteriosclerosis. 1986; 6: 131-138Crossref PubMed Google Scholar, 7Frostegard J Ulfgren AK Nyberg P Hedin U Swedenborg J Andersson U Hansson GK Cytokine expression in advanced human atherosclerotic plaques: dominance of pro-inflammatory (Th1) and macrophage-stimulating cytokines.Atherosclerosis. 1999; 145: 33-43Abstract Full Text Full Text PDF PubMed Scopus (828) Google Scholar Vascular smooth muscle cells (SMCs) also have a prominent role in plaque growth and stability through their continuous production of fibrous tissue. Indeed, inability of these cells to keep their phenotype contributes largely to plaque rupture and disease progression.8Owens GK Kumar MS Wamhoff BR Molecular regulation of vascular smooth muscle cell differentiation in development and disease.Physiol Rev. 2004; 84: 767-801Crossref PubMed Scopus (2557) Google Scholar The Epstein-Barr virus (EBV)-induced gene 3 (Ebi3) is a member of the interleukin (IL)-12 heterodimeric cytokine family. It was initially discovered as an EBV-induced cellular gene in infected human B lymphocytes. It encodes a soluble hematopoietin receptor-type protein of 34-kd that lacks a membrane-anchoring motif that bears structural similarities to the IL-12β chain (also known as p40).9Devergne O Hummel M Koeppen H Le Beau MM Nathanson EC Kieff E Birkenbach M A novel interleukin-12 p40-related protein induced by latent Epstein-Barr virus infection in B lymphocytes.J Virol. 1996; 70: 1143-1153Crossref PubMed Google Scholar, 10Devergne O Birkenbach M Kieff E Epstein-Barr virus-induced gene 3 and the p35 subunit of interleukin 12 form a novel heterodimeric hematopoietin.Proc Natl Acad Sci USA. 1997; 94: 12041-12046Crossref PubMed Scopus (275) Google Scholar It associates as a heterodimer with either IL-27α (also known as p28) to yield IL-27,11Pflanz S Timans JC Cheung J Rosales R Kanzler H Gilbert J Hibbert L Churakova T Travis M Vaisberg E Blumenschein WM Mattson JD Wagner JL To W Zurawski S McClanahan TK Gorman DM Bazan JF de Waal Malefyt R Rennick D Kastelein RA IL-27, a heterodimeric cytokine composed of EBI3 and p28 protein, induces proliferation of naive CD4(+) T cells.Immunity. 2002; 16: 779-790Abstract Full Text Full Text PDF PubMed Scopus (1157) Google Scholar or with IL-12α/p35 (also known as p35), to form the recently described IL-35.12Collison LW Workman CJ Kuo TT Boyd K Wang Y Vignali KM Cross R Sehy D Blumberg RS Vignali DA The inhibitory cytokine IL-35 contributes to regulatory T-cell function.Nature. 2007; 450: 566-569Crossref PubMed Scopus (1471) Google Scholar, 13Niedbala W Wei XQ Cai B Hueber AJ Leung BP McInnes IB Liew FY IL-35 is a novel cytokine with therapeutic effects against collagen-induced arthritis through the expansion of regulatory T cells and suppression of Th17 cells.Eur J Immunol. 2007; 37: 3021-3029Crossref PubMed Scopus (487) Google Scholar Although Ebi3 expression has been shown to be largely restricted to cells of the myeloid lineage,11Pflanz S Timans JC Cheung J Rosales R Kanzler H Gilbert J Hibbert L Churakova T Travis M Vaisberg E Blumenschein WM Mattson JD Wagner JL To W Zurawski S McClanahan TK Gorman DM Bazan JF de Waal Malefyt R Rennick D Kastelein RA IL-27, a heterodimeric cytokine composed of EBI3 and p28 protein, induces proliferation of naive CD4(+) T cells.Immunity. 2002; 16: 779-790Abstract Full Text Full Text PDF PubMed Scopus (1157) Google Scholar it has been also described in placental syncytiotrophoblasts,14Devergne O Coulomb-L'Hermine A Capel F Moussa M Capron F Expression of Epstein-Barr virus-induced gene 3, an interleukin-12 p40-related molecule, throughout human pregnancy: involvement of syncytiotrophoblasts and extravillous trophoblasts.Am J Pathol. 2001; 159: 1763-1776Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar endothelial cells,15Kempe S Kestler H Lasar A Wirth T NF-kappaB controls the global pro-inflammatory response in endothelial cells: evidence for the regulation of a pro-atherogenic program.Nucleic Acids Res. 2005; 33: 5308-5319Crossref PubMed Scopus (220) Google Scholar plasma cells,16Larousserie F Pflanz S Coulomb-L'Hermine A Brousse N Kastelein R Devergne O Expression of IL-27 in human Th1-associated granulomatous diseases.J Pathol. 2004; 202: 164-171Crossref PubMed Scopus (115) Google Scholar Hodgkin and Reed-Sternberg lymphoma cells,17Niedobitek G Pazolt D Teichmann M Devergne O Frequent expression of the Epstein-Barr virus (EBV)-induced gene. EBI3, an IL-12 p40-related cytokine, in Hodgkin and Reed-Sternberg cells.J Pathol. 2002; 198: 310-316Crossref PubMed Scopus (81) Google Scholar and in B cell lymphomas.18Larousserie F Bardel E Coulomb L'Hermine A Canioni D Brousse N Kastelein RA Devergne O Variable expression of Epstein-Barr virus-induced gene 3 during normal B-cell differentiation and among B-cell lymphomas.J Pathol. 2006; 209: 360-368Crossref PubMed Scopus (24) Google ScholarEbi3 deficient mice (Ebi3−/−) do not show overt autoimmunity or inflammatory disease. In fact, these mice have impaired type 2 T helper (TH2)19Nieuwenhuis EE Neurath MF Corazza N Iijima H Trgovcich J Wirtz S Glickman J Bailey D Yoshida M Galle PR Kronenberg M Birkenbach M Blumberg RS Disruption of T helper 2-immune responses in Epstein-Barr virus-induced gene 3-deficient mice.Proc Natl Acad Sci USA. 2002; 99: 16951-16956Crossref PubMed Scopus (151) Google Scholar as well as delayed TH1 immune responses.20Zahn S Wirtz S Birkenbach M Blumberg RS Neurath MF von Stebut E Impaired Th1 responses in mice deficient in Epstein-Barr virus-induced gene 3 and challenged with physiological doses of Leishmania major.Eur J Immunol. 2005; 35: 1106-1112Crossref PubMed Scopus (48) Google Scholar Recent evidence suggested that Ebi3−/− mice are protected in a concanavalin-A-induced hepatitis model, showing reduced interferon (IFN) γ production and STAT1/T-bet activation on concanavalin-A administration.21Siebler J Wirtz S Frenzel C Schuchmann M Lohse AW Galle PR Neurath MF Cutting edge: a key pathogenic role of IL-27 in T cell-mediated hepatitis.J Immunol. 2008; 180: 30-33Crossref PubMed Scopus (40) Google Scholar In addition, important immunomodulatory functions have been described to both Ebi3 heterodimers, IL-27 and IL-35. By inducing T-bet through STAT1-dependent and -independent mechanisms, IL-27 augments T cell proliferation and regulates the expression of IFNγ and IL-12Rβ2 in naive T cells, priming these cells to respond to IL-12 and to differentiate into TH1 cells.11Pflanz S Timans JC Cheung J Rosales R Kanzler H Gilbert J Hibbert L Churakova T Travis M Vaisberg E Blumenschein WM Mattson JD Wagner JL To W Zurawski S McClanahan TK Gorman DM Bazan JF de Waal Malefyt R Rennick D Kastelein RA IL-27, a heterodimeric cytokine composed of EBI3 and p28 protein, induces proliferation of naive CD4(+) T cells.Immunity. 2002; 16: 779-790Abstract Full Text Full Text PDF PubMed Scopus (1157) Google Scholar, 22Lucas S Ghilardi N Li J de Sauvage FJ IL-27 regulates IL-12 responsiveness of naive CD4+ T cells through Stat1-dependent and -independent mechanisms.Proc Natl Acad Sci USA. 2003; 100: 15047-15052Crossref PubMed Scopus (387) Google Scholar, 23Hamano S Himeno K Miyazaki Y Ishii K Yamanaka A Takeda A Zhang M Hisaeda H Mak TW Yoshimura A Yoshida H WSX-1 is required for resistance to Trypanosoma cruzi infection by regulation of proinflammatory cytokine production.Immunity. 2003; 19: 657-667Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar Experiments using IL-27 receptor -deficient mice (WSX-1−/−) revealed that IL-27 plays an important role in TH1 differentiation during infection with some intracellular pathogens.24Chen Q Ghilardi N Wang H Baker T Xie MH Gurney A Grewal IS de Sauvage FJ Development of Th1-type immune responses requires the type I cytokine receptor TCCR.Nature. 2000; 407: 916-920Crossref PubMed Scopus (336) Google Scholar, 25Yoshida H Hamano S Senaldi G Covey T Faggioni R Mu S Xia M Wakeham AC Nishina H Potter J Saris CJ Mak TW WSX-1 is required for the initiation of Th1 responses and resistance to L. major infection.Immunity. 2001; 15: 569-578Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar However, other studies using infectious and autoimmune inflammatory models have shown that WSX-1 −/− mice develop aberrantly elevated TH1 and TH2 responses, suggesting an important role of IL-27 as attenuator of the immune/inflammatory response.23Hamano S Himeno K Miyazaki Y Ishii K Yamanaka A Takeda A Zhang M Hisaeda H Mak TW Yoshimura A Yoshida H WSX-1 is required for resistance to Trypanosoma cruzi infection by regulation of proinflammatory cytokine production.Immunity. 2003; 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175: 2401-2407PubMed Google Scholar Furthermore, it was recently demonstrated that IL-27 can suppress TH17 immune responses to extracellular bacteria as well as in autoimmune models.30Stumhofer JS Laurence A Wilson EH Huang E Tato CM Johnson LM Villarino AV Huang Q Yoshimura A Sehy D Saris CJ O'Shea JJ Hennighausen L Ernst M Hunter CA Interleukin 27 negatively regulates the development of interleukin 17-producing T helper cells during chronic inflammation of the central nervous system.Nat Immunol. 2006; 7: 937-945Crossref PubMed Scopus (774) Google Scholar, 31Batten M Li J Yi S Kljavin NM Danilenko DM Lucas S Lee J de Sauvage FJ Ghilardi N Interleukin 27 limits autoimmune encephalomyelitis by suppressing the development of interleukin 17-producing T cells.Nat Immunol. 2006; 7: 929-936Crossref PubMed Scopus (697) Google Scholar, 32Fitzgerald DC Ciric B Touil T Harle H Grammatikopolou J Das Sarma J Gran B Zhang GX Rostami A Suppressive effect of IL-27 on encephalitogenic Th17 cells and the effector phase of experimental autoimmune encephalomyelitis.J Immunol. 2007; 179: 3268-3275PubMed Google Scholar The heterodimeric hematopoietin p35/Ebi3 described in human and mouse10Devergne O Birkenbach M Kieff E Epstein-Barr virus-induced gene 3 and the p35 subunit of interleukin 12 form a novel heterodimeric hematopoietin.Proc Natl Acad Sci USA. 1997; 94: 12041-12046Crossref PubMed Scopus (275) Google Scholar was recently shown to be constitutively secreted by regulatory T cells.12Collison LW Workman CJ Kuo TT Boyd K Wang Y Vignali KM Cross R Sehy D Blumberg RS Vignali DA The inhibitory cytokine IL-35 contributes to regulatory T-cell function.Nature. 2007; 450: 566-569Crossref PubMed Scopus (1471) Google Scholar, 13Niedbala W Wei XQ Cai B Hueber AJ Leung BP McInnes IB Liew FY IL-35 is a novel cytokine with therapeutic effects against collagen-induced arthritis through the expansion of regulatory T cells and suppression of Th17 cells.Eur J Immunol. 2007; 37: 3021-3029Crossref PubMed Scopus (487) Google Scholar Designated as IL-35, this cytokine is required for regulatory T cells full suppressive functions and represents a novel anti-inflammatory mechanism suppressing the immune response through expansion of regulatory T cells and inhibition of TH17 cell development.12Collison LW Workman CJ Kuo TT Boyd K Wang Y Vignali KM Cross R Sehy D Blumberg RS Vignali DA The inhibitory cytokine IL-35 contributes to regulatory T-cell function.Nature. 2007; 450: 566-569Crossref PubMed Scopus (1471) Google Scholar, 13Niedbala W Wei XQ Cai B Hueber AJ Leung BP McInnes IB Liew FY IL-35 is a novel cytokine with therapeutic effects against collagen-induced arthritis through the expansion of regulatory T cells and suppression of Th17 cells.Eur J Immunol. 2007; 37: 3021-3029Crossref PubMed Scopus (487) Google Scholar To date nothing is known about the role of Ebi3 and Ebi3-related novel cytokines in cardiovascular disease. Our previous finding that Ebi3 is strongly up-regulated by TNFα/NF-κB signaling in endothelial cells15Kempe S Kestler H Lasar A Wirth T NF-kappaB controls the global pro-inflammatory response in endothelial cells: evidence for the regulation of a pro-atherogenic program.Nucleic Acids Res. 2005; 33: 5308-5319Crossref PubMed Scopus (220) Google Scholar prompted us to investigate the potential role of Ebi3 in atherosclerosis. We therefore analyzed the in situ expression of Ebi3, as well as of IL-27α/p28 and IL-12α/p35 subunits, in human atherosclerotic plaque samples. By immunohistochemistry we found Ebi3 expression in atherosclerotic lesions. Expression of Ebi3 was observed in regions of vascular smooth muscle cells, endothelial cells and macrophages. In addition, expression of IL-27α/p28 and IL-12α/p35 subunits was also found in vascular smooth muscle cells. Experiments with primary aortic smooth muscle cells (AoSMCs) confirmed inducible Ebi3 expression and provided insights in its regulation. These data suggest a possible role for Ebi3 and Ebi3-related cytokines in atherogenesis that requires further investigation. Plaque samples were obtained from 12 nondiabetic patients with symptomatic high-grade stenosis of the carotid artery. Nondiabetic state was assessed by a negative history for diabetes mellitus, no treatment with antidiabetic drugs. Patients scheduled for carotid endarterectomy and were recruited at the Department of Thoracic and Vascular Surgery, University of Ulm, Germany.33Meisner F Walcher D Gizard F Kapfer X Huber R Noak A Sunder-Plassmann L Bach H Haug C Bachem M Stojakovic T Marz W Hombach V Koenig W Staels B Marx N Effect of rosiglitazone treatment on plaque inflammation and collagen content in nondiabetic patients: data from a randomized placebo-controlled trial.Arterioscler Thromb Vasc Biol. 2006; 26: 845-850Crossref PubMed Scopus (61) Google Scholar After surgery, plaque samples were immediately frozen (Tissue Tek, O.C.T.) and stored in liquid nitrogen. For immunohistochemical staining, serial cryostat sections (10 μm thick) of carotid specimens were probed with the following antibodies: anti-human CD68 antibody (Dako), anti-human CD31 antibody (Dako), anti-human α-actin antibody (Dako), anti-human Ebi3 (2G4H6 mAb, 33 kd), anti-human IL27/p28 (Abcam), and anti-human IL-12p35 (Abcam). For negative controls, isotype-matched IgG at similar concentrations were used. Sections were incubated with the respective biotinylated secondary antibody (Dako) followed by avidin-biotin-peroxidase complex (Vector Laboratories). Antibody binding was visualized with 3-amino-9-ethyl carbazole. Sections were counterstained with Gill’s hematoxylin (Sigma). Primary human AoSMCs (Cambrex) were cultured in smooth muscle cells basal medium (SmGM-2, Clonetics) supplemented with 5% fetal bovine serum. Experiments were performed with cells between passages 4 and 7 in serum-free media (Dulbecco’s modified Eagle’s medium, Gibco). Cells were stimulated with human recombinant TNFα (10 ng/ml, a gift from Dr. Adolf, Boehringer-Ingelheim, Vienna) or IFNγ (1000 U/ml, Peprotech Inc.). Rosiglitazone (BRL) (10 μmol/L, Cayman Chemical) was added 1 hour prior the cytokine treatment. Total RNA was isolated from106 cells using the High Pure RNA isolation kit (Roche). First-strand cDNA was synthesized using 2 μg of RNA and M-MLV reverse transcriptase (Promega). Quantitative real-time PCR was performed using the QuantiTect SYBR Green PCR kit (Qiagen) and the Roche light cycler system. Primer sequences used were: human Ebi3 forward 5′-CAGCTTCGTGCCTTTCATAA-3′, reverse 5′-CTCCCACTGCACCTGTAGC-3′ (annealing temperature 59°C); human IL27α/p28 forward 5′-ATCTCACCTGCCAGGAGTGAA-3′, reverse 5′-TGAAGCGTGGTGGAGATGAAG-3′ (annealing temperature 60°C); human IL12α/p35 forward 5′-TTCACCACTCCCAAAACCTGC-3′, reverse 5′-GAGGCCAGGCAACTCCCATTAG-3′ (annealing temperature 57°C); and human β-actin forward 5′-TGTGGCATCCACGAAACTAC-3′, reverse 5′-GGAGCAATGATCTTGATCTTCA-3′ (annealing temperature 60°C). Quantitative RT-PCR was analyzed using the ΔΔCt method with efficiency correction according to Pfaffl.34Pfaffl MW A new mathematical model for relative quantification in real-time RT-PCR.Nucleic Acids Res. 2001; 29: e45Crossref PubMed Scopus (25161) Google Scholar Different treatments did not affect control β-actin transcript levels, as shown in Supplemental Figure S1 at . β-Actin housekeeping gene stability was determined from four endogenous reference genes (βactin, HPRT, PBGD, and RPL13a) using geNorm software35Vandesompele J De Preter K Pattyn F Poppe B Van Roy N De Paepe A Speleman F Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.Genome Biol. 2002; 3 (research 0034.1-0034.11)Crossref PubMed Google Scholar and is shown in Supplemental Figure S1 at . Cells (approximately 5 × 105) were lysed on ice in 50 μl of 2X concentrated Laemmli sample buffer. Proteins were denatured, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 10% polyacrylamide gel, and electroblotted onto a nitrocellulose membrane (Millipore). Membranes were probed with anti-human Ebi3antibody (2G4H6 mAb, 33 kd) and anti-human actin antibody (44 kd, Sigma). Signals were visualized using the SuperSignal West Dura extended-duration substrate (Pierce). Chromatin immunoprecipitation was performed as described previously.36Ushmorov A Ritz O Hummel M Leithauser F Moller P Stein H Wirth T Epigenetic silencing of the immunoglobulin heavy-chain gene in classical Hodgkin lymphoma-derived cell lines contributes to the loss of immunoglobulin expression.Blood. 2004; 104: 3326-3334Crossref PubMed Scopus (87) Google Scholar AoSMCs were pretreated with 10 μmol/L BRL for 1 hour and stimulated with 10 ng/ml TNFα for 24 hours in the presence or absence of BRL. One microgram of rabbit anti-p65/RelA antibody (Santa Cruz Biotechnology) and rabbit anti-PLCγ1 antibody (Santa Cruz Biotechnology) were used. Samples were analyzed by PCR using specific primers for the NF-κB binding region in Ebi-3 and IκBα human promoters. Primer sequences used for human IκBα promoter were: forward 5′-GACGACCCCAATTCAAATCG-3′, reverse 5′-GTCAGGCTCGGGGAATTTCC-3′ (annealing temperature 57°C); and for human Ebi3 promoter: forward 5′-CTCTGTCTCCCTCCATGTCC-3′, reverse 5′-TTCCCAGCACAGCATGTC-3′ (annealing temperature 59°C). Given the immunomodulatory functions of Ebi3-containing cytokines we wanted to examine Ebi3 expression in human arteriosclerotic lesions. Therefore, we performed immunohistochemical analyses in human endarterectomy specimens obtained from nondiabetic patients with high grade stenosis (n = 12). Ebi3-expression was detectable in almost all advanced lesions analyzed (n = 10/12). Serial sections stained with endothelial cell CD-31 surface marker, and the macrophage CD-68 surface marker revealed Ebi3 positive expression in regions of the these cell types (see Supplemental Figures S2 and S3 at ). Interestingly, we also observed Ebi3 staining in areas staining positive for the vascular smooth muscle cell marker α-actin (Figure 1, Figure 2). Staining of Ebi3 in vascular SMC positive areas was seen in the intima and media (Figure 1, A, C, F; Figure 2, A, C, F). Staining of parallel sections demonstrated a weak IL-27α/p28 subunit positive staining in atheroma vascular SMCs (Figure 1, B, D, E). Furthermore, a strong IL-12α/p35 positive staining was observed spread throughout the atheroma samples and apparent co-expression with Ebi3 was also found in atheroma vascular SMCs (Figure 2, B, D, E). Staining of sections with isotype-matched control antibodies showed no immunoreactivity, thus affirming the specificity of the detected signals (Figure 1, G–I; Figure 2, G–I). These data suggest the expression of Ebi3 in vascular SMCs either as homodimers or as IL-27 and IL-35 heterodimers.Figure 2Immunohistochemical analysis of Ebi3 expression in atheroma lesions. Ebi3 and p35 colocalization; sections from human carotid endarterectomy specimen 2. An overview detailing the magnified region is shown to the left. A and D: Ebi3 staining. B and E: IL-12α/p35 staining. C and F: SMC α-actin staining. G–I: Control stainings with matched IgG antibodies. L, lumen. The indicated objective magnifications (×20, ×40) were used.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Given the expression of Ebi3 in vascular smooth muscle cells in human arteriosclerotic lesions, we next analyzed whether proinflammatory stimulation of human primary AoSMCs would result in a transcriptional up-regulation of the Ebi3 gene. To this end, cells were stimulated with either TNFα or IFNγ for various time points, and expression of Ebi3 was evaluated by real time quantitative RT-PCR. Both cytokines up-regulated Ebi3 mRNA expression reaching a peak of induction after 24 hours of stimulation (Figure 3, A and B). TNFα stimulation resulted in a 17-fold induction compared with approximately fivefold induction by IFNγ. To determine whether the increased mRNA levels would result in a corresponding induction of Ebi3 protein expression, we analyzed AoSMCs by Western blot analysis after treatment with TNFα for 24 hours. This analysis revealed a strong induction of Ebi3 protein expression in these stimulated SMCs (Figure 3C). Given that both TNFα and IFNγ alone induced Ebi3 expression, we asked whether combined stimulation with these two proinflammatory cytokines might further increase Ebi3 expression. As seen in Figure 3D, we found a synergistic induction of Ebi3 expression 24 hours after stimulating AoSMCs with both cytokines. Since our immunohistochemical data in human atheroma revealed a co-expression of Ebi3 with IL-27α/p28 and IL-12α/p35, we analyzed whether the expression of p28 and p35 subunits could also be induced in smooth muscle cells by either of the two inflammatory cytokines. These analyses revealed that IL-27α/p28 expression was up-regulated by TNFα treatment and to a lower extent in IFNγ-treated AoSMCs, while synergistic up-regulation by cotreatment with both cytokines was observed (Figure 3D). Similarly, expression analyses of the p35 subunit showed a low expression in AoSMCs that was significantly induced by TNFα and IFNγ stimulation. The level of synergistic up-regulation was less pronounced for p35 (Figure 3D). In previous work we showed that treatment with the PPARγ agonist rosiglitazone of nondiabetic patients with symptomatic carotid artery stenosis resulted in a reduction of vascular inflammation and in an increase of the plaque collagen content, leading to a more stable atherosclerotic lesion.33Meisner F Walcher D Gizard F Kapfer X Huber R Noak A Sunder-Plassmann L Bach H Haug C Bachem M Stojakovic T Marz W Hombach V Koenig W Staels B Marx N Effect of rosiglitazone treatment on plaque inflammation and collagen content in nondiabetic patients: data from a randomized placebo-controlled trial.Arterioscler Thromb Vasc Biol. 2006; 26: 845-850Crossref PubMed Scopus (61) Google Scholar In addition, there is ample evidence that PPARγ agonists interfere with the NF-κB signaling pathway, which is strongly induced in proinflammatory situations.37Pascual G Fong AL Ogawa S Gamliel A Li AC Perissi V Rose DW Willson TM Rosenfeld MG Glass CK A SUMOylation-dependent pathway mediates transrepression of inflammatory response genes by PPAR-gamma.Nature. 2005; 437: 759-763Crossref PubMed Scopus (1018) Google Scholar We therefore addressed the question as to whether TNFα- and IFNγ- induced expression of Ebi3 might be modulated by activation of PPARγ. Indeed, we found that rosiglitazone treatment of AoSMCs resulted in a significant reduction of Ebi3 up-regulation on TNFα stimulation but had no effect on IFNγ-induced Ebi3 expression (Figure 4, A and B). To further test whether rosiglitazone might even attenuate the synergistic activation of Ebi3 expression by TNFα and IFNγ costimulation, we analyzed protein extracts of AoSMCs that were treated with rosiglitazone and stimulated for 24 hours with both TNFα and IFNγ. The PPARγ activator rosiglitazone markedly reduced the synergistic activation of Ebi3 expression by both proinflammatory cytokines (Figure 4C). The stimulation by TNFα/IFNγ and inhibition by rosiglitazone suggested that the transcriptional up-regulation of Ebi3 could be mediated by NF-κB. Recently it was shown
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